Drug release from antithrombogenic multi-layer coated stent

ABSTRACT

A stent having a multi-layered coating adhered to its surface which can prevent restenosis and thrombosis at the implant site. The stent coating is comprised of two layers. The first layer is a polymeric coating with one or more biologically active agent(s) dispersed therein. The second layer is comprised of a hydrophobic heparinized polymer that inhibits blood coagulation and provides a hydrophilic surface for reducing the friction between stent and lesion site. In preferred embodiments of the invention, the multi-layered stent is effective in deterring restenosis and thrombosis at the implant site. In these same preferred embodiments, the multi-layered stent is capable of reducing the burst release of the biologically active agents from the first layer and sustaining a release of an effective amount of these agents for a relatively extended period of time. Methods of applying the multi-layered coating to the stent surface are also part of this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No.10/262,432, filed Sep. 30, 2002, now U.S. Pat. No. 6,702,850, which ishereby incorporated in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] This invention relates to coated stents for carrying biologicallyactive agents to provide localized treatment at the implant site andmethods of applying stent coatings. In particular, this inventionrelates to antithrombogenic and antirestenotic stents having amulti-layered coating, wherein the first or inner layer is formed from apolymer and one or more biologically active agents, and a second orouter layer is formed from an antithrombogenic heparinized polymer. Thisinvention also relates to methods of applying a multi-layer coating overthe surface of a stent and methods of using such a coated stent.

[0004] Atherogenic arterial narrowing and thrombosis are two potentiallyfatal, related conditions that have been identified as leading killersby various health organizations in the United States and throughout theworld. Stenosis refers to the narrowing or constriction of a vessel,which is usually due to the buildup of fat, cholesterol, and othersubstances over time. In severe cases, stenosis can completely clog avessel. Thrombosis is the formation or presence of a blood clot inside ablood vessel or cavity of the heart. The clot is usually formed by anaggregation of blood factors, primarily platelets and fibrin, withentrapment of cellular elements. Thrombosis, like stenosis, frequentlycauses vascular obstruction at the point of its formation.

[0005] One approach to clearing an artery that has been constricted orclogged due to stenosis is percutaneous transluminal coronaryangioplasty (PTCA) or balloon coronary angioplasty. In this procedure, aballoon catheter is inserted and expanded in the constricted portion ofthe vessel for clearing the blockage. About one-third of patients whoundergo PTCA suffer from restenosis, the renarrowing of the widenedsegment, within about six months of the procedure. Restenosed arteriesmay have to undergo another angioplasty.

[0006] Restenosis can be inhibited by a common procedure that consistsof inserting a stent into the effected region of the artery instead of,or along with, angioplasty. A stent is a tube made of metal or plastic,which can have either solid walls or mesh walls. Most stents in use aremetallic and are either self-expanding or balloon-expandable. Thedecision to undergo a stent insertion procedure depends on certainfeatures of the arterial stenosis. These include the size of the arteryand the location of the stenosis. The function of the stent is tobuttress the artery that has recently been widened using angioplasty,or, if no angioplasty was used, the stent is used to prevent elasticrecoil of the artery. Stents are typically implanted via a catheter. Inthe case of a balloon-expandable stent, the stent is collapsed to asmall diameter and slid over a balloon catheter. The catheter is thenmaneuvered through the patient's vasculature to the site of the lesionor the area that was recently widened. Once in position, the stent isexpanded and locked in place. The stent stays in the artery permanently,holds it open, improves blood flow through the artery, and relievessymptoms (usually chest pain).

[0007] Stents are not 100% effective in preventing restenosis at theimplant site. Restenosis can occur over the length of the stent and/orpast the ends of the stent. Physicians have recently employed new typesof stents that are coated with a thin polymer film loaded with a drugthat inhibits smooth cell proliferation. The coating is applied to thestent prior to insertion into the artery using methods well known in theart, such as a solvent evaporation technique. The solvent evaporationtechnique entails mixing the polymer and drug in a solvent. The solutioncomprising polymer, drug, and solvent can then be applied to the surfaceof the stent by either dipping or spraying. The stent is then subjectedto a drying process, during which the solvent is evaporated, and thepolymeric material, with the drug dispersed therein, forms a thin filmlayer on the stent.

[0008] The release mechanism of the drug from the polymeric materialsdepends on the nature of the polymeric material and the drug to beincorporated. The drug diffuses through the polymer to the polymer-fluidinterface and then into the fluid. Release can also occur throughdegradation of the polymeric material. The degradation of the polymericmaterial occurs through hydrolysis, which erodes the polymer into thefluid and hence releases the drug into the fluid as well.

[0009] An important consideration in using coated stents is the releaserate of the drug from the coating. It is desirable that an effectivetherapeutic amount of the drug be released from the stent for thelongest period of time possible. Burst release, a high release rateimmediately following implantation, is undesirable and a persistentproblem. While typically not harmful to the patient, a burst release“wastes” the limited supply of the drug by releasing several times theeffective amount required and shortens the duration of the releaseperiod. Several techniques have been developed in an attempt to reduceburst release. For example, U.S. Pat. No. 6,258,121 B1 to Yang et al.discloses a method of altering the release rate by blending two polymerswith differing release rates and incorporating them into a single layer.

[0010] Heparin, generally derived from swine intestine, is a substancethat is well known for its anticoagulation ability. It is known in theart to apply a thin polymer coating loaded with heparin onto the surfaceof a stent using the solvent evaporation technique. For example, U.S.Pat. No. 5,837,313 to Ding et al. describes a method of preparing aheparin coating composition.

[0011] In view of the foregoing, it will be appreciated that thedevelopment of a stent having a multi-layered coating, where one layercomprises a thin film of polymeric material with a biologically activeagent dispersed therein, and a second layer is disposed over the firstlayer where the second layer comprises a hydrophobic heparinizedpolymer, would be a significant advance in the art. It will also beappreciated that the current invention inhibits both restenosis andthrombosis, and can be effective in delivering a wide range of othertherapeutic agents to the implant site over a relatively extended periodof time.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention provides a stent having a multi-layeredcoating comprising at least two layers disposed one on top of the otherfor inhibiting restenosis and thrombosis through the delivery ofbiologically active agents over a sustained period of time. The firstlayer comprises a polymeric material with a biologically active agentdispersed therein, and the second layer comprises a hydrophobicheparinized polymer having effective anticoagulation characteristics.This invention also provides several methods for applying multiple innerlayers of a coating onto a stent, with the hydrophobic heparinizedpolymer being applied as the outer and final layer of the coating.

[0013] The first or sub-layer is prepared by mixing a polymeric materialand a biologically active agent with a solvent, thereby forming ahomogeneous solution. The polymeric material can be selected from a widerange of synthetic materials, but in one illustrative embodiment,polyacrylic acid is used. The biologically active agent is selecteddepending on the desired therapeutic results. For example, an anticancerdrug and/or antiinflammatory drug can be used. By way of furtherexample, if an inhibitor of smooth cell proliferation is desired,echinomycin or paclitaxel can be used. Once prepared, the solution canbe applied to the stent through a dipping or spraying process. Duringdrying, the solvent evaporates, and a thin layer of the polymericmaterial loaded with the biologically active agent is left coated overthe stent. It should be noted that the current invention is not limitedto just one inner layer or biologically active agent per layer. It iswithin the scope of this invention to add one or more distinctbiologically active agents to each layer and/or have more than one innerlayer loaded with a biologically active agent.

[0014] The second or outer layer is an antithrombogenic heparinizedpolymer applied to the stent over the inner layer using, for example, adipping process. The antithrombogenic heparinized polymer coating isprepared by bonding multifunctional macromolecules, such as polyacrylicacid, and hydrophobic materials, such as octadecylamine, with heparin.In one illustrative embodiment of the invention, the hydrophobicmaterial has more than one reactive functional group and under 100 mg/mlwater solubility after being combined with a macromolecule. The stent isthen dipped in the hydrophobic heparinized polymer, which has been mixedwith a solvent. After drying, the solvent evaporates and the hydrophobicheparinized polymer forms a thin film over the first layer.

[0015] The coated stent is inserted into the afflicted vessel, such as acoronary artery, using an appropriate procedure that depends on thestent style. Once in place, the stent structure will hold the vesselopen. The biologically active agent will be released from the firstlayer, thereby providing the desired therapeutic result, such asinhibiting smooth cell proliferation. The antithrombogenic heparinizedpolymer prevents blood coagulation around the stent, thus inhibitingthrombosis and subacute stent thrombosis. In addition, theantithrombogenic heparinized polymer layer reduces or prevents the burstrelease of the biologically active agent from the first layer, therebyallowing the release to occur over a relatively extended period of time.

[0016] Accordingly, it is an advantage of this invention to provide astent capable of inhibiting restenosis and thrombosis at theimplantation site.

[0017] It is also an advantage of the invention to provide a stenthaving a multi-layered coating for delivering biologically active agentsthat inhibit smooth cell proliferation and blood clots.

[0018] It is still another advantage of the invention to provide a stenthaving a multi-layered coating having an outer layer comprising anantithrombogenic heparinized polymer.

[0019] It is still another advantage of the invention to provide a stenthaving a multi-layered coating for delivering a wide range oftherapeutic agents over a relatively extended period of time andmitigating the burst release of the biologically active agent from theinner layer or layers.

[0020] It is yet another advantage of the invention to provide a methodfor applying a multi-layered coating to the surface of a stent.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0021]FIG. 1 is a cross-sectional representation of a fragmentaryportion of a stent showing the first and second layers of the stentcoating.

[0022]FIG. 2(a) is a scanning electron microscope (SEM) image from arough surface of a bare stent, magnified 2,000 times.

[0023]FIG. 2(b) is an SEM image of a smooth surface of a stent with afirst-layer coating that has been applied by the dipping method,magnified 2,000 times.

[0024]FIG. 2(c) is an SEM image of a smooth surface of a multi-layeredcoated stent, where both the first and second layers were applied by thedipping method, magnified 2,000 times.

[0025]FIG. 3(a) is an SEM image of a stent surface coated by aspray-coating method, magnified 50 times.

[0026]FIG. 3(b) is an SEM image of the stent surface of FIG. 3(a),magnified 2000 times.

[0027]FIG. 4 shows three stent surfaces after they have undergone awhole blood test; Stent A is the bare stent, Stent B is the first-layercoated stent, and Stent C is the multi-layer coated stent.

[0028]FIG. 5(a) is an SEM image of a bare stent surface after a plateletadhesion test, magnified 3000 times.

[0029]FIG. 5(b) is an SEM image of a first-layer coated stent after aplatelet adhesion test, magnified 3000 times.

[0030]FIG. 5(c) is an SEM image of a multi-layer coated stent after aplatelet adhesion test, magnified 3000 times.

[0031]FIG. 6(a) shows five distinctly coated strips after 14 days insideof male Sprague-Dawley rats, each of the strips exhibiting differentlevels of inflammation.

[0032]FIG. 6(b) shows five distinctly coated strips as in FIG. 6(a)after 30 days inside of male Sprague-Dawley rats, each of the stripsexhibiting different levels of inflammation.

[0033]FIG. 7(a) illustrates the percentage of paclitaxel released forboth a single layer coating and a multi-layer coating over a nine-dayperiod.

[0034]FIG. 7(b) illustrates the amount of paclitaxel released per unitarea for both a single layer coating and a multi-layer coating over anine-day period.

[0035]FIG. 8(a) illustrates the percentage of echinomycin anddexamethasone released over a nine day period for a multi-layeredcoating:

[0036]FIG. 8(b) illustrates the amount of echinomycin and dexamethasonereleased per unit area over a nine day period for a multi-layeredcoating.

[0037]FIG. 9(a) shows a cross-section of a blood vessel in which a barestent was implanted.

[0038]FIG. 9(b) shows a cross-section of a blood vessel in which amulti-layer coated stent was implanted with a 0.1% loading ofechinomycin in the first layer.

[0039]FIG. 9(c) shows a cross-section of a blood vessel in which amulti-layer coated stent was implanted with a 1% loading of echinomycinin the first layer.

[0040]FIG. 9(d) shows a cross-section of a blood vessel in which amulti-layer coated stent was implanted with a 5% loading of echinomycinin the first layer.

[0041]FIG. 10 illustrates a restenosis evaluation by the comparison ofintima versus media (intima/media).

DETAILED DESCRIPTION

[0042] Before the present stent having a multilayered coat and methodsfor using and preparing a stent having a multi-layered coating aredisclosed and described, it is to be understood that this invention isnot limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

[0043] The publications and other reference materials referred to hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

[0044] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a biologically active agent” includes a mixtureof two or more of such agents, reference to “an inhibitor” includesreference to one or more of such inhibitors, and reference to “thesolvent” includes reference to a mixture of two or more solvents.

[0045] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0046] As used herein, “stent” means a tube of metal or plastic that isinserted into a vessel or passage to keep the lumen open and preventclosure due to a stricture or external compression.

[0047] As used herein, “biologically active agent” means a drug or othersubstance that has therapeutic value to a living organism includingwithout limitation antithrombotics, anticoagulants, antiplatelet agents,thrombolytics, antiproliferatives, anti-inflammatories, agents thatinhibit restenosis, smooth muscle cell inhibitors, antibiotics, and thelike, and mixtures thereof.

[0048] Illustrative anticancer drugs include acivicin, aclarubicin,acodazole, acronycine, adozelesin, alanosine, aldesleukin, allopurinolsodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine,androgens, anguidine, aphidicolin glycinate, asaley, asparaginase,5-azacitidine, azathioprine, Bacillus calmette-guerin (BCG), Baker'sAntifol (soluble), beta-2′-deoxythioguanosine, bisantrene hcl, bleomycinsulfate, busulfan, buthionine sulfoximine, BWA 773U82, BW 502U83-HCl, BW7U85 mesylate, ceracemide, carbetimer, carboplatin, carmustine,chlorambucil, chloroquinoxaline- sulfonamide, chlorozotocin, chromomycinA3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum,CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine,cytembena, dabis maleate, dacarbazine, dactinomycin, daunorubicin HCl,deazauridine, dexrazoxane, dianhydrogalactitol, diaziquone,dibromodulcitol, didemnin B, diethyldithiocarbamate, diglycoaldehyde,dihydro-5-azacytidine, doxorubicin, echinomycin, edatrexate, edelfosine,eflomithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin,estramustine phosphate, estrogens, etanidazole, ethiofos, etoposide,fadrazole, fazarabine, fenretinide, filgrastim, finasteride. flavoneacetic acid, floxuridine, fludarabine phosphate, 5-fluorouracil,Fluosol®, flutamide, gallium nitrate, gemcitabine, goserelin acetate,hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazinesulfate, 4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl,ifosfamide, interferon alfa, interferon beta, interferon gamma,interleukin-1 alpha and beta, interleukin-3, interleukin-4,interleukin-6, 4-ipomeanol, iproplatin, isotretinoin, leucovorincalcium, leuprolide acetate, levamisole, liposomal daunorubicin,liposome encapsulated doxorubicin, lomustine, lonidamine, maytansine,mechlorethamine hydrochloride, melphalan, menogaril, merbarone,6-mercaptopurine, mesna, methanol extraction residue of Bacilluscalmette-guerin, methotrexate, N-methylformamide, mifepristone,mitoguazone, mitomycin-C, mitotane, mitoxantrone hydrochloride,monocyte/macrophage colony-stimulating factor, nabilone, nafoxidine,neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin,paclitaxel, pala, pentostatin, piperazinedione, pipobroman, pirarubicin,piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimersodium, prednimustine, procarbazine, progestins, pyrazofurin, razoxane,sargramostim, semustine, spirogermanium, spiromustine, streptonigrin,streptozocin, sulofenur, suramin sodium, tamoxifen, taxotere, tegafur,teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa,thymidine injection, tiazofurin, topotecan, toremifene, tretinoin,trifluoperazine hydrochloride, trifluridine, trimetrexate, tumornecrosis factor, uracil mustard, vinblastine sulfate, vincristinesulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, andmixtures thereof.

[0049] Illustrative antiinflammatory drugs include classic non-steroidalanti-inflammatory drugs (NSAIDS), such as aspirin, diclofenac,indomethacin, sulindac, ketoprofen, flurbiprofen, ibuprofen, naproxen,piroxicam, tenoxicam, tolmetin, ketorolac, oxaprosin, mefenamic acid,fenoprofen, nambumetone (relafen), acetaminophen (Tylenol®), andmixtures thereof; COX-2 inhibitors, such as nimesulide, NS-398,flosulid, L-745337, celecoxib, rofecoxib, SC-57666, DuP-697, parecoxibsodium, JTE-522, valdecoxib, SC-58125, etoricoxib, RS-57067, L-748780,L-761066, APHS, etodolac, meloxicam, S-2474, and mixtures thereof;glucocorticoids, such as hydrocortisone, cortisone, prednisone,prednisolone, methylprednisolone, meprednisone, triamcinolone,paramethasone, fluprednisolone, betamethasone, dexamethasone,fludrocortisone, desoxycorticosterone, and mixtures thereof; andmixtures thereof.

[0050] As used herein, “polymer” means a macromolecule made of repeatingmonomer units or protomers.

[0051] As used herein, “macromolecule” means synthetic macromolecules,proteins, biopolymers and other molecules with a molecular weighttypically greater than 1000.

[0052] As used herein, “antithrombogenic heparinized polymer,”“hydrophobic heparinized polymer,” and similar terms mean hydrophobicmulticomponent heparin conjugates as described International PatentApplication No. PCT/KR00/01255 (WO 02/34312 A1), co-owned with thepresent invention, which is incorporated herein by reference in itsentirety. These hydrophobic heparinized polymers comprise a conjugatecomprising heparin, a macromolecular component, and a hydrophobiccomponent.

[0053] As used herein, “effective amount” means an amount ofpharmacologically active agent that is nontoxic but sufficient toprovide the desired local or systemic effect and performance at areasonable benefit/risk ratio attending any medical treatment.

[0054] The present invention relates to an antithrombogenic stent havinga multi-layered coating. In an illustrative embodiment, the first layercomprises a polymeric film loaded with a biologically active agent thatprevents smooth cell proliferation, such as echinomycin. Illustrativepolymers that can be used for making the polymeric film includepolyurethanes, polyethylene terephthalate (PET), PLLA-poly-glycolic acid(PGA) copolymer (PLGA), polycaprolactone (PCL).poly-(hydroxybutyrate/hydroxyvalerate) copolymer (PHBV),poly(vinylpyrrolidone) (PVP), polytetrafluoroethylene (PTFE, Teflon™),poly(2-hydroxyethylmethacrylate) (poly-HEMA), poly(etherurethane urea),silicones, acrylics, epoxides, polyesters, urethanes, parlenes,polyphosphazene polymers, fluoropolymers, polyamides, polyolefins, andmixtures thereof. The second layer comprises a hydrophobic heparinizedpolymer with strong anticoagulation properties. The second layer of thehydrophobic heparinized polymer also has the effect of preventing aburst release of the biologically active agent dispersed in the firstlayer—resulting in a relatively longer release period of thebiologically active agent. It should also be understood that the firstlayer can contain more than one biologically active agent.

[0055] The style and composition of the stent may comprise anybiocompatible material having the ability to support a diseased vessel.In general, it is preferred to use a metal stent, such as thosemanufactured from stainless steel, gold, titanium or the like, butplastic or other appropriate materials may be used. In one preferredembodiment, the stent is a Palmz-Schatz stent manufactured by CordisCorp. (Miami, Fla.). The stent may be self expanding or balloonexpanding. It is preferred that the coating substantially cover theentire stent surface, but it is within the scope of this invention tohave the coating cover only a portion of the stent. It is also to beunderstood that any substrate, medical device, or part thereof havingcontact with organic fluid, or the like, may also be coated.

[0056] The application of the first layer is accomplished through asolvent evaporation process or some other known method. The solventevaporation process entails combining the polymeric material and thebiologically active agent with a solvent, such as tetrahydrofuran (THF),which are then mixed by stirring to form a mixture. An illustrativepolymeric material of the first layer comprises polyurethane and anillustrative biologically active agent comprises echinomycin. Themixture is then applied to the surface of the stent by either: (1)spraying the solution onto the stent; or (2) dipping the stent into thesolution. After the mixture has been applied, the stent is subjected toa drying process, during which, the solvent evaporates and the polymericmaterial and biologically active agent form a thin film on the stent. Inanother illustrative embodiment, more than one biologically active agentcan be added to the first layer.

[0057] The second layer of the stent coating comprises anantithrombogenic heparinized polymer. Antithrombogenic heparinizedpolymers are soluble only in organic solvents and are insoluble inwater. Antithrombogenic heparin polymers are produced by binding heparinto macromolecules and hydrophobic materials.

[0058] Illustrative macromolecules include synthetic macromolecules,proteins, biopolymers, and mixtures thereof. Illustrative syntheticmacromolecules include polydienes, polyalkenes, polyacetylenes,polyacrylic acid and its derivatives, poly α-substituted acrylic acidand its derivatives, polyvinyl ethers, polyvinylalcohol, polyvinylhalides, polystyrene and its derivatives, polyoxides, polyethers,polyesters, polycarbonates, polyamides, polyamino acids, polyureas,polyurethanes, polyimines, polysulfides, polyphosphates, polysiloxanes,polysilsesquioxanes, polyheterocyclics, cellulose and its derivatives,and polysaccharides and their copolymers or derivatives. Illustrativeproteins that can be used according to the present invention includeprotamine, polylysine, polyaspartic acid, polyglutamic acid, andderivatives and copolymers thereof. Illustrative biopolymers that can beused according to the present invention include polysaccharides,gelatin, collagen, alginate, hyaluronic acid, alginic acid, carrageenan,chondroitin, pectin, chitosan, and derivatives and copolymers thereof Inan illustrative embodiment, the antithrombogenic heparin polymer isprepared by the following steps. First, the macromolecules are activatedby using N,N-dicyclohexylcarbodiimide hydrochloride (DDC) or4-p-azidosalicylamido-butylamine (ASBA). Second, binding heparin,recombinant heparin, heparin derivatives or heparin analogues (having apreferred weight of 1,000-1,000,000 daltons) to the macromolecules.While covalent bonds can formed between the heparin and macromoleculesby using the hydroxyl group, amine group, thiol group or azide group, itis preferred to use the amine group. Lastly, the hydrophobic materialsare bound with the functional groups of the macromolecule which hasalready been combined with heparin. While it is understood that anyhydrophobic material that has a functional group can be used, it ispreferred to use octadecylamine, alkanoic amine, bile acids, sterols, oralkanoic acids.

[0059] Once the hydrophobic heparinized polymer has been prepared, thesecond layer is applied directly over the first layer using the solventevaporation method or other appropriate method. The hydrophobicheparinized polymer is readied for application by combining it with asolvent, such as cyclohexane, thereby forming an aqueous solution havingthe hydrophobic heparinized polymer suspended therein. Theantithrombogenic heparinized polymer and solvent solution is thenapplied to the stent using a dipping process. The solvent is evaporatedfrom the stent during a drying process; leaving a thin film of thehydrophobic heparinized polymer over the first layer.

[0060] The antithrombogenic heparinized polymer layer inhibitscoagulation at the implant site. In addition, the second layer inhibitsor prevents a burst release of the biologically active agent from thefirst layer. The second layer also serves to extend the release periodof the biologically active agent from the first layer, therebylengthening the treatment time.

EXAMPLE 1

[0061] Preparation of a Multi-Layer Coating on a Stent by a Dip-CoatingMethod. A coating solution for the first layer was prepared by combiningand agitating a polymer, biologically active agent and THF, untilthoroughly mixed. The polymer selected was polyurethane and had aconcentration of 3 wt %. The biologically active agent was paclitaxeland had a concentration range of 0 to 20 wt %. Prior to applying thefirst layer, the stent's surface was prepared and cleaned by washing itwith methanol and drying it in a vacuum drier for approximately 30minutes. Once dry, the cleaned stent was fully immersed into the firstcoating solution and dried at room temperature for approximately 5 hoursin a beaker saturated with THF. This dipping/drying process was repeated5 times. After the fifth repetition, the stent was dried at roomtemperature for about 1 hour in a vacuum drier.

[0062] The second layer coating solution was prepared by mixing thehydrophobic heparinized polymer, 0.1 to 20 wt %, in cyclohexane. Thestent was then dipped into the heparinized polymer solution and dried atroom temperature for 1 hour, followed by drying in a vacuum drier atroom temperature for 6 hours. The two-layer coating as applied on thesurface of a stent is illustrated in FIG. 1.

EXAMPLE 2

[0063] Preparation of a Multi-Layered Coating on a Stent by aSpray-Coating Method. A coating solution for the first layer and stentwere prepared as described in Example 1. The prepared first-layersolution was then sprayed on the cleaned stent for approximately 10minutes and dried at room temperature. This spraying/drying process wasrepeated 10 times, after which the stent was dried in a vacuum drier forapproximately 1 hour. The second coating layer, the hydrophobicheparinized polymer solution, was applied as described in Example 1. Thetwo-layer coating as applied on the surface of a stent is illustrated inFIG. 1.

EXAMPLE 3

[0064] Preparation of a Multi-Layer Coated Stent Loaded with TwoBiologically Active Agents. The first layer coating solution wasprepared by combining and agitating polyurethane, paclitaxel,dexamethasone in THF until thoroughly mixed. The loading amounts of thebiologically active agents were 0 to 20 wt %, respectively. The stentwas cleaned as described in Example 1. The prepared solution was sprayedon the stent for approximately 10 minutes and dried at room temperature.After the spray process was repeated for 10 times, the stent was driedunder vacuum for 1 hour. The second layer, a hydrophobic heparinizedpolymer solution, was applied as described in Example 1.

EXAMPLE 4

[0065] Morphology of the Multi-Layer Coated Stent. The surface of bothan uncoated and coated stent were examined under a scanning electronmicroscope at varying powers of magnification. The surface of theuncoated metal stent was observed to have a very rough appearance asshown in FIG. 2(a). The surface of the stent was then observed after theapplication of both the first layer and the second layer. In both cases,the coated surface was observed to be relatively much smoother than thatof the bare stent. This held true irregardless of whether a dipping or aspaying method was used to apply the first layer. This is shown in FIGS.2(b) and 2(c), and FIGS. 3(a) and 3(b), respectively.

EXAMPLE 5

[0066] Blood Compatibility of the Multi-Layer Coated Stent—Whole BloodTest. Three stainless steel stents, Stents A, B, and C, were providedfor this test. The Stent A was left bare and had no coating applied. TheStent B had a single layer coating of polyurethane, with paclitaxelloaded therein, applied to the stent surface. Finally, the Stent C hadthe first-layer coating as on the second stent plus a top layer of thehydrophobic heparinized polymer. All three stents were dipped in freshrabbit blood for a period of approximately 3 minutes. After removal, thestents were examined to determine the level of thrombus formation on thestent surfaces. The Stent A was observed to have a relatively high levelof thrombus formation and blood coagulation on its surface. Stent B wasobserved to have a decreased amount of thrombus formation and bloodcoagulation, when compared to the first stent. The Stent C exhibited areduced amount of thrombus formation when compared to the second stent.FIG. 4 shows a picture of all three stents and the varying degrees ofthrombosis.

EXAMPLE 6

[0067] Blood Compatibility of Multi-Layer Coated Stent—Platelet AdhesionTest. Fresh rabbit blood was mixed with 3.8 wt % sodium citrate solutionat a 9:1 ratio concentration. The blood was then placed in a centrifugeand spun at 2,000 rpm for 10 minutes at 5° C. to isolate the plateletsin a plasma. The plasma platelet concentration was manipulated by addingplatelet-poor plasma, spun at 4,000 RPM, until a concentration level of3×10⁵/μl was obtained. Three stainless steel stents were then preparedas in Example 5. The stents were incubated in the prepared plasma at 37°C. for approximately 1 hour. After removal, the stents were washed threetimes with a PBS solution. The stents then underwent a platelet fixationprocess which consisted of incubating the stents in 2.5% glutaraldehydefor 4 hours. Upon completion of platelet fixation, the stents werewashed in 50%, 80%, and 100% ethanol aqueous solutions. After the secondwashing, the samples were freeze dried for 6 hours. The stents were thenexamined under a scanning electron microscope to determine the plateletconcentration present on each of the stent's surface. The bare stentshowed an uniform distribution of platelet formation on its surface asshown in FIG. 5(a). The second stent, with a single polyurethane layer,and the third stent, with the multi-layer coating, showed a decrease of80% and 90%, respectively, in the level of platelet adhesions as shownin FIG. 5(b) and FIG. 5(c).

EXAMPLE 7

[0068] Evaluation of Inflammation of Multi-Layer Coated Stent. A numberof stainless steel strips of five varying types were prepared withdifferent compositions of surface coating. As shown in the followingchart, Strip Types A, B, C, D, and E, all had either no coating, aone-layer coating, or a two-layer coating. The strips were prepared forimplantation into male Sprague-Dawley rat. Strip Type First Layer SecondLayer A None None B polyurethane loaded with paclitaxel (20 wt %) None Cpolyurethane loaded with paclitaxel (20 wt %) hydrophobic heparinizedpolymer D polyurethane loaded with paclitaxel (20 wt %) None anddexamethasone (5 wt %) E polyurethane loaded with paclitaxel (20 wt %)hydrophobic and dexamethasone (5 wt %) heparinized polymer

[0069] The rats, weighing between 200-300 g, were chosen at random. Therats were first anesthetized with diethyl ether gas and secured to anoperating table. One of the five types of steel strips was inserted intothe back of each rat through an incision made by a scalpel. The stripswere then recovered after either 14 or 30 days. The strips wererecovered by anesthetizing the rats again with diethyl ether and thensurgically removing a region right below where the strip had beeninserted as well as the regions of tissue where it appeared thatrestenosis has occurred. After removal, the strip and tissue were washedwith a PBS buffer solution. The tissue was then fixed with a 4%formaldehyde solution. Each strip was then visually examined todetermine the level of restenosis, if any, that had developed relativeto the other strips.

[0070] After 14 days, the bare strip types, Strip Type A, showed severerestenosis. For those strips that had had paclitaxel loaded inpolyurethane applied as primary coating, Strip Type B, a considerabledegree of restenosis was observed. On the other hand, strips that had asecond coating of a hydrophobic heparinized polymer, Strip Type C, hadalmost no restenosis. Moreover, when the specimen that had received astrip whose first layer of polyurethane had been loaded withdexamethasone, an anti-inflammatory drug, and paclitaxel, Strip Type D,those specimens showed almost no restenosis. The same held true forspecimens that had an additional second layer of a hydrophobicheparinized polymer, Strip Type E. These results are shown in FIG. 6(a).

[0071] After 30 days, severe restenosis was observed on both theuncoated strips, Strip Type A, and on the single layered strips loadedwith paclitaxel, Strip Type B. In strips loaded with paclitaxel anddexamethasone, Strip Type D, restenosis was also observed. However, whena hydrophobic heparinized polymer was applied as an outer layer, StripTypes C and E, no restenosis could be observed. This results are shownin FIG. 6(b).

EXAMPLE 8

[0072] Elution of Paclitaxel from the Multi-Layer Coated Film. Theamount of paclitaxel eluted from a single layer polyurethane coating ona stainless steel sample was compared to a multi-layered coated sample.Both samples were incubated separately in a buffer solution at 37° C.The eluted paclitaxel was measured at 4, 8, 12, 24, 36, 48, 60, 144, 216hours by an extraction method comprising the following steps. First, thesolution was extracted by using 6 ml DCM per 100ml buffer solution withstrong agitation for about 15 seconds. Next, the solution in DCM partwas separated and dried under nitrogen gas. Finally, the extractedpaclitaxel was dissolved in 1 ml acetonitrile and measured by HPLC.

[0073] As shown in FIGS. 7(a) and 7(b), the elution rate of paclitaxelfrom the multi-layer coated film showed a decreased burst release and amore sustained release pattern, when compared to single-layer coatedfilm.

EXAMPLE 9

[0074] Elution of Echinomycin from the Multi-Layer Coated Stent. Theelution of echinomycin from a multi-layered coated stent was evaluated.Multi-layer coated stents were prepared using the method of Example 2,except that echinomycin was loaded into the first layer and notpaclitaxel. The loading amount of echinomycin was 3 wt %, and the spraycoating process was repeated 10 times. The second layer was formed by adip coating with 1 wt % hydrophobic heparinized polymer solution. Asshown in FIGS. 8(a) and 8(b), the elution rate of paclitaxel from themulti-layer coated film showed a decreased burst release and a moresustained release pattern, when compared to single-layer coated film.

EXAMPLE 10

[0075] Elution of Dexamethasone from the Multi-Layer Coated Stent.Dexamethasone was loaded into the polyurethane first layer of a coatedstent at 5 wt %. A second layer was applied by a dipping method in a 1wt % hydrophobic heparinized polymer solution as described in Example 1.The elution rate of dexamethasone from the coated stent was thenmeasured. As shown in FIGS. 8(a) and 8(b), the dexamethasone showed adecreased burst release, and the elution rate was higher than that ofechinomycin as in Example 9.

EXAMPLE 11

[0076] Evaluation of Restenosis for Multi-Layer Coated Stent—StentPreparation and Animal Selection. Five groups of three stents each werefirst prepared, the stents of each group having the same coating (or nocoating), and each group having a distinct coating, varying both in thenumber of layers and drug composition and/or concentration. Each grouphad one of the following coatings: a polymer control stent, a barestent, and three multi-layer coated stents having a hydrophobicheparinized polymer outer layer and echinomycin loaded at 0.1% wt, 1% wtor 5% wt. Fifteen pigs were then selected and divided at random intogroups containing three pigs each. The average pig weighed 23 kg andprior to the experiment, the pigs were all kept in the same conditionsand fed an experimental feed devoid of lipids. The pigs were alsoadministered 300 mg/day of aspirin through their feed.

EXAMPLE 12

[0077] Evaluation of Restenosis for Multi-Layer Coated Stent—Methods ofExperiment. Each pig was systemically anesthetized with an injection ofketamine (22 mg/kg) and prepared for surgery. Next, an incision was madein the front of the neck at the midline exposing the carotid artery. Adose of heparin(300U/kg) was injected into the artery of the pig at thistime. An 8 French artery guide-wire was then inserted into the carotidartery through a small incision in the arterial wall. A guide catheterwas then inserted and maneuvered to, and inside of, the left and rightcoronary artery. An appropriate site on the right coronary artery wasselected with the use of a coronary artery angiography.

[0078] The appropriate stent was attached to a balloon catheter having aballoon capable of expanding to 10-20% larger than the diameter of thecoronary artery. The balloon catheter was maneuvered to the sitepreviously selected in the coronary artery and the balloon was inflatedto its maximum size for 30 seconds at 4-12 atmospheric pressure tointentionally damage the coronary artery. After the balloon wasdeflated, the stent remained at the site. It should be noted, that inorder to block the coronary artery spasm following the blood vesseldamage, nitroglycerin (200 μg) was continuously administered into thecoronary artery through the guiding catheter. After the operation, acoronary artery angiography was conducted to observe the degree ofdamage to the coronary artery and the patency of the blood flow. Theartery guide-wire was then removed and the slit in the carotid arterywas ligated.

[0079] After 28 days, the pigs were again anesthetized and a guide-wireinserted as before. A dose of heparin (300 U/kg) was again injected viaguide-wire into the artery. After confirming the patency of the bloodvessels in the coronary artery, lethal amounts of pentothal andpotassium chloride were injected via the guide catheter to induceeuthanasia. The pig's heart was then removed through the thorax. Theheart was then subjected to a perfusion-fixation procedure. Beforesacrificing the animals, follow-up coronary angiography using OEC (GEmedical, USA) was employed to determine the size of blood vessels andpictures taken before and after blood vessel damage were evaluated inorder to determine the location and degree of arterial narrowing of thestented coronary segment.

EXAMPLE 13

[0080] Evaluation of Restenosis for Multi-Layer CoatedStent—Histological Evaluation. The damaged portion of the artery alongwith an additional 2 cm region around the damaged site was removed fromthe heart. The specimen containing the stent was fixed using theEmbedding System (Technovit 7100, Kulzer, Germany). The specimen wasthen sliced into thin pieces with the use of a microtome equipped with atungsten blade. Each slice was dyed with hematoxylin-eosin and elasticVan Gieson.

[0081] Each slice was then studied under a microscope. The slices wereevaluated using the Schwartz scale. A quantitative and morphologicalanalysis of the slices was conducted. In particular, the lumen area,internal elastic lamina area and external elastic area, intimal area,medial area, and the I/M ratio were determined. FIGS. 9(a)-9(d) and 10show the results and the I/M ratio. The results confirmed that themulti-layered stent whose first layer had been loaded with 1 wt %echinomycin showed a significantly reduced level of neointimal tissuevolume at 28 days when compared to the other stents and especially thebare stent.

The subject matter claimed is:
 1. A method for preparing an article ofmanufacture comprising a stent and a coating disposed thereon, thecoating comprising a first layer and a second layer, the first layercomprising a polymer film with a biologically active agent dispersedtherein, and the second layer comprising an antithrombogenic heparinizedpolymer, the method comprising: cleaning the stent with a washing agent,preparing the first layer by combining the polymer and biologicallyactive agent with a solvent, thereby forming a polymer and biologicallyactive agent mixture and applying the mixture to the stent, preparingthe second layer by combining the hydrophobic heparinized polymer with asolvent and applying the second layer by immersing the stent in thehydrophobic heparinized polymer and solvent solution and then drying thestent.
 2. The method of claim 1 further comprising adding a secondbiologically active agent to the polymer and biologically active agentmixture.
 3. The method of claim 1 wherein applying the first layercoating comprises dipping the stent into the polymer and biologicallyactive agent mixture.
 4. The method of claim 1 wherein applying thefirst layer coating comprises spraying the polymer and biologicallyactive agent mixture onto the stent.
 5. A method for preventing burstrelease of a biologically active agent dispersed in a thin film polymerlayer on a stent comprising applying a second layer over the firstlayer; said second layer being comprised of a hydrophobic heparinizedpolymer.
 6. A method for inhibiting thrombosis in a medical devicehaving a surface in contact with an organic fluid comprising coating thesurface of the medical device with an antithrombogenic heparinizedpolymer layer.
 7. The method of claim 6 further comprising applying alowermost coating, said lowermost coating disposed under the hydrophobicheparinized polymer layer and comprising an polymer having abiologically active agent dispersed therein.